Narayanaswamy Mathangi, Griffiths William J, Howard Philip W, Thurston David E
School of Pharmacy, University of London, London WCIN 1AX, UK.
Anal Biochem. 2008 Mar 1;374(1):173-81. doi: 10.1016/j.ab.2007.11.018. Epub 2007 Nov 17.
The main method of evaluating the DNA interstrand cross-linking ability of cancer chemotherapeutic agents in naked DNA currently involves the electrophoresis of relatively long radiolabeled duplex DNA fragments (typically approximately 2000 bp) on neutral gels after incubation with the agent of interest. Denaturation by heating is carried out prior to loading, and a neutral gel allows reannealing of cross-linked DNA. To avoid the use of radioactivity we have developed a new method based on ion pair reversed phase liquid chromatography (RPLC) and mass spectrometry (MS) that allows characterization and quantitation of drug-DNA interstrand cross-links formed within short oligonucleotide duplexes (i.e., 12 bp). Advantages of this assay include rapid throughput, as compared to electrophoretic methods, and the use of readily available short nonradiolabeled oligonucleotides of any sequence, thereby facilitating investigation of sequence selectivity. A further advantage is that all species separated by the chromatographic process can be positively identified by MS. Using this new method, we have investigated the rate of DNA cross-linking and sequence selectivity of the interstrand cross-linking agent SJG-136, a pyrrolobenzodiazepine (PBD) dimer currently in phase I clinical trials. The assay was found to be sufficiently sensitive and selective to allow separation of the unbound and drug-bound oligonucleotide species by high-performance liquid chromatography (HPLC) and to allow positive identification of these individual species by MS. A further benefit, as compared with electrophoretic methods, is that kinetic information can be obtained and compared for different binding sequences.
目前，评估癌症化疗药物在裸露DNA中形成链间交联能力的主要方法是，将相对较长的放射性标记双链DNA片段（通常约为2000 bp）与感兴趣的药物孵育后，在中性凝胶上进行电泳。上样前需通过加热使其变性，中性凝胶可使交联的DNA重新退火。为避免使用放射性物质，我们开发了一种基于离子对反相液相色谱（RPLC）和质谱（MS）的新方法，该方法可对短寡核苷酸双链体（即12 bp）内形成的药物 - DNA链间交联进行表征和定量。与电泳方法相比，该检测方法的优点包括通量高，且可使用任何序列的易于获得的短非放射性标记寡核苷酸，从而便于研究序列选择性。另一个优点是，通过色谱过程分离的所有物种都可以通过质谱进行阳性鉴定。使用这种新方法，我们研究了链间交联剂SJG - 136（一种目前正处于I期临床试验阶段的吡咯并苯并二氮杂卓（PBD）二聚体）的DNA交联速率和序列选择性。结果发现该检测方法具有足够的灵敏度和选择性，能够通过高效液相色谱（HPLC）分离未结合和药物结合的寡核苷酸物种，并通过质谱对这些单个物种进行阳性鉴定。与电泳方法相比，另一个好处是可以获得不同结合序列的动力学信息并进行比较。
